Extraction of cell-bound protein from bordetella

ABSTRACT

PCT No. PCT/EP94/00597 Sec. 371 Date Oct. 23, 1995 Sec. 102(e) Date Oct. 23, 1995 PCT Filed Feb. 28, 1994 PCT Pub. No. WO94/20538 PCT Pub. Date Sep. 15, 1994The present invention discloses a process for extracting a cell-bound protein of bacterial origin, useful in acellular vaccines, comprising contacting a suspension of the cell-bound protein with a flocculating agent prior to heat treatment.

This application is a 371 of PCT/EP94/00597 filed Feb. 28, 1994 whichclaims priority to foreign application 9304398.0 filed in Great Britainon Mar. 4, 1993.

The present invention relates to a novel process for the isolation ofcell proteins having utility as antigenic factors of component oracellular vaccines. In particular, the invention relates to a novelprocess for the extraction of outer membrane proteins of bacterialorganisms, for example the outer membrane protein of Bordetellapertussis, which has a molecular weight of approximately 69,000 Daltonsand is generally referred to as the 69 kD protein of Bordetellapertussis, or pertactin.

Whooping cough, or pertussis, is a highly-infectious disease whichprimarily affects children. In addition to causing respiratorycomplications, whooping cough may result in nerve damage and a highincidence of mortality, particularly in children from low socioeconomicgroups and in newborn infants who do not possess maternal anti-pertussisantibodies. The etiologic agent of pertussis is the Gram-negativecoccobacillus, Bordetella pertussis. The bacteria are believed to invadethe respiratory tract and induce a toxic state which remains even aftertheir disappearance, several days later.

The disease is currently controlled through immunisation with "wholecell" vaccine prepared by growing the Bordetella pertussis organism infermenters and then inactivating the resulting cells by heat treatmentand/or addition of chemical agents. Although the World HealthOrganisation presently recommends the immunisation of infants to preventthe incidence and spread of pertussis, concern has arisen over thereported adverse events resulting from various vaccine formulations. Theconsequent reduced usage of conventional B. pertussis vaccine hasresulted in an increase in the incidence of pertussis infections. Theneed for a pertussis vaccine which avoids the reported adverse eventsfrom whole cell vaccine is recognised. Considerable research effort hasaccordingly been put into the development of an efficaceous acellularvaccine comprising a small number of highly-purified antigenicproteinaceous components.

A number of antigens have been proposed as acellular vaccine components,including for example lymphocytosis promoting factor (LPF), also knownas histamine sensitising factor, islet activating protein or, morecommonly, pertussis toxin (PT); filamentous hemagglutinin (FHA); andfimbrial agglutinogens.

A further potential antigen is one of the outer membrane proteins of thebacterium, having a molecular weight of approximately 69,000 Daltons(pertactin) found in all virulent strains of B. pertussis. The B.pertussis 69 kD protein is immunologically-related to similar proteinshaving slight differences in electrophoretic mobility which are producedby the human pathogen B. parapertussis and the animal pathogen B.bronchiseptica. Although the 69 kD protein is secreted in relativelysmall amounts into the fermentation broth during the cultivation of B.pertussis, the majority of it is found attached to the cell membrane,from which it may be readily extracted. Published procedures for theextraction and purification of the 69 kD protein do not however allowfor large-scale commercial production of a highly-purified and stableantigen.

EP-0 162 639 describes acid-glycine extraction of the cells followed byseveral purification steps culminating in an affinity chromatographyseparation using a specific monoclonal antibody. The protein obtainedhas been reported to have both poor stability and adenylate cyclaseactivity, and the downstream purification procedure is not suitable forlarge-scale production.

Brennan et al. (Infection and Immunity 56, 3189-3195, 1988) describe afurther method whereby protein is released from cells by heat treatmentand a protein extract is obtained which is purified by chromatography onfetuin-Sepharose and a monoclonal antibody affinity column.

U.S. Ser. No. 7/308,864 describes extraction and purification involvingheat treatment and centrifugation, followed by DEAE-Sepharoseion-exchange chromatography and protein-specific, dye-ligand gelchromatography. This method avoids the use of expensive monoclonalantibodies but is nevertheless regarded as inappropriate for large-scaleproduction. One particular problem which has been identified is thesmall quantity of 69 kD protein released into solution as a percentageof the total protein released after the heat treatment. EP-A-0 437 687sets out to improve the efficiency of release of 69 kD protein from theculture broth by using a repetitive extraction process involving aplurality of extraction steps in series.

The present invention overcomes the problem of inefficient proteinrecovery by providing a process which enhances the yield of proteinreleased from the microbial cells after a single extraction step andalso obviates the requirement for subsequent centrifugation to removethe cell debris. Furthermore, the process of the present invention hasthe additional advantage that it effectively eliminates most of the highmolecular weight endotoxins (lipo-polysaccharides) which are present inthe culture broth after fermentation and are further released intosolution when the membrane proteins are extracted by application ofheat. Heat treatment of microbial cell suspensions followingfermentation is an important step in the isolation of proteins, forexample outer membrane proteins, which are poorly secreted by themicrobes into the culture broth during fermentation. The 69 kD proteinof B. pertussis is typical of such outer membrane proteins which aresecreted in insufficient quantity to warrant direct isolation from thebroth supernatant at production scale. In this respect it differs fromthe other antigen candidates for an acellular pertussis vaccine,pertussis toxin (PT) and filamentous haemagglutinin (FHA).

Heat treatment involves heating the cell suspension in buffer at, forexample, 60° C. for approximately one hour, during which time the outermembrane proteins slough off the cell membrane into solution. Thesolution is not however a free-flowing liquid but is largely aglutinous, jelly-like mass comprising dead cells and the contentstherefrom, which can be separated (by for example, filtration orcentrifugation) from the free-flowing liquid on a small scale (5-10 mlaliquots) only with difficulty. This heavily-hydrated jelly frequentlycontains more than 50% of the original liquid volume and would, ifdiscarded, considerably reduce the yield of 69 kD protein. Coarsefiltration is moreover unsatisfactory because the jelly immediatelyblocks the filter and effectively prevents passage of liquid.

The present invention provides an effective method for eliminating theformation of a jelly-like mass during the heat treatment. It has beenfound that addition of a flocculating agent to the cell suspension priorto heat treatment causes an intense flocculation of the cells to takeplace. The flocculated mass of cells can be readily separated from thesuspending broth, and may be washed to remove unwanted broth componentsbefore heat treatment. The dead cells re-flocculate after heattreatment, leaving a substantially cell-free supernatant containing themajority of soluble protein. This procedure gives a high-percentagerecovery of the desired protein, in solution, for downstreampurification.

The present invention confers yet further advantage in that it canprovide recovered protein which has to be subjected to less-stringentdownstream purification because most of the high molecular weightendotoxins are eliminated from the protein-containing solution obtainedafter heat treatment together with the flocculated mass of dead cells.The possibility therefore exists for reducing the number of downstreampurification steps required to achieve the requisite standard of proteinpurity demanded for prophylactic and therapeutic applications.

The present invention accordingly provides for a process for extractingcell-bound protein of bacterial origin comprising contacting asuspension of the cell-bound protein with a flocculating agent.

A wide range of flocculating agents well known in the art may beemployed in the process of the invention to improve the handlingqualities of the cell suspension following heat treatment. Preferredflocculating agents for use in the present invention are materialsembodying divalent cations. Suitable divalent cations for use in theinvention are made available from salts of barium, calcium andstrontium, for example halide salts such as barium chloride, calciumchloride or strontium chloride, preferably barium chloride.

The flocculating agent is suitably brought into contact with asuspension of cells under controlled-pH conditions and the liquid volumeis adjusted by addition of appropriate buffer. After mixing theflocculated cells are allowed to settle. The flocculated cells aresuitably collected by sedimentation (or centrifugation) and may bewashed with saline or buffer before exposure to heat treatment.

The extraction process of the invention, when used in conjunction withfurther downstream processing, gives rise to 69 kD protein from B.pertussis with a very high level of purity. It has, for example, nodetectable levels of PT or heat-labile toxin, and endotoxin levels arereduced to nanograms per mg of protein. In addition, the protein hasnone of the enzyme activity, in particular adenylate cyclase activity,associated with earlier production methods.

In a preferred embodiment of the invention, 69 kD protein is produced ina fermentation broth or culture of B. pertussis. Suitable strains foruse in the invention are described and are readily available incommercial collections such as the American Type Culture Collection,Rockville, Md., USA. Preferred strains are those which are capable ofgrowing in liquid culture media, and of producing high yields of 69 kDprotein.

Examples of strains which may be employed include, without limitation,B. pertussis phase I, B. pertussis phase II, B. pertussis phase I CS, B.pertussis Tohama, B. pertussis strain 185-30, B. pertussis strain 18323,B. pertussis strain 134, B. pertussis strain 509, B. pertussis strainWellcome 28, and Office of Biologics B. pertussis strain 165. Apreferred strain for use in the present invention is B. pertussis phaseI, Tohama, which is available from the Institute of Fermentation, Osaka,Japan under accession number IFO-14073.

For use in the present invention the selected B. pertussis strain can begrown in a variety of ways known to persons skilled in the art. Variouscultivation methods are known which employ different cultivation steps,and liquid or solid media, depending on the quantity and origin orconservation methods of the seed culture. However, any known method willsuffice for use in the present invention which provides an inoculum of aconventionally-acceptable size for large-scale production.

A suitable medium for growth of a B. pertussis inoculum may be selectedby any person skilled in the art. Suitable media include, withoutlimitation, Gengou medium (EP-A-0 077 646); the media described in N.Andorn et al. (Appl. Microbiol. Biotechnol., 28, 356-360, 1988) andreferences cited therein; Stainer-Scholte medium (J. Gen. Microbiol.,63, 211-220, 1971); modified Stainer-Scholte medium described in A.Imaizumi et al (Infect. Immun., 41, 3, 1138-1143, 1983 and J. Microbiol.Methods, 2, 339-347, 1984); Verway medium (U.S. Pat. No. 4,784,589);synthetic medium B2 (P. Van Hemert; Prog. Indust. Microbiol.; (Bull, M.J. ed.), Vol 13, p. 151, Elsevier Sci., Amsterdam (1977)) or describedmodifications thereof.

For growth of B. pertussis culture which is a starting material of thepresent invention, an inoculum is added to a suitable liquid medium andfermentation is conducted employing conventional fermentation methodsand fermenter designs known in the art. Persons skilled in the art willappreciate that different results may be obtained depending upon theselection of a particular combination of conventional fermenter design,fermentation medium, method and parameters. Preferred combinations foruse in the present invention are those suitable for use in large-scaleproduction. Examples of such combinations of methods, designs and mediaare exemplified in EP-A-0 077 646 and preferably EP-A-0 121 249 andEP-A-0 239 504.

After completion of the fermentation, the B. pertussis fermentationbroth is suitably processed to remove the antigenic factors PT and FHAwhich are secreted directly into the broth, for example by the methoddescribed in EP-A-0 427 462 by adsorption on hydroxyapatite, themethodology of which is incorporated herein by reference.

The residual microbial suspension containing the cell-bound 69 kDprotein is then treated with a flocculating agent according to thepresent invention before being subjected to heat treatment in order toeffect release of the protein.

It has been found that residual endotoxin levels may be minimised bycontrolling the pH of the supernatant. By routine experimentation, theoptimum pH for any chosen flocculating agent may be selected. Thus, thepH of the supernatant is suitably adjusted to between 4 and 10,preferably between pH 8.5 and 9.5, by addition of base, either before orafter addition of an aqueous solution of for example a halide salt ofbarium, calcium or strontium, preferably barium chloride. The liquidvolume may be adjusted by addition of buffer, suitably TRIS buffer.Suitably mixing is carried out for approximately 5 minutes and theresulting slurry is allowed to flocculate over a period of approximately1 hour. The supernatant containing the unused components of the growthmedium (salts, aminoacids, minerals, etc.) and other proteinaceousmaterials and endotoxins released into the broth during fermentation maythen be separated and discarded, and the flocculate may be washed withfurther buffer or saline. Alternatively, the flocculated cells may beseparated by centrifugation before washing.

Since the high concentration of salts, amino acids and minerals presentin the fermentation broth may interfere with subsequent purificationsteps, the flocculation/washing process may be repeated as many times asis necessary to obtain a slurry of flocculated cells in which theconcentration of halide salt has been reduced to an acceptable level,for example, approximately 0.02% w/v.

The slurry of flocculated cells is then subjected to heat treatment at atemperature of approximately 60° C. for 15-60 minutes during which timethe 69 kD protein is liberated into the liquid suspension and thebacteria are killed. After cooling, for example at approximately 4° C.for an appropriate duration, preferably overnight, the dead cell mass isremoved, for example by decantation or centrifugation, and thesupernatant is filtered and stored under sterile conditions at reducedtemperature, for example at about 4° C.

The 69 kD protein may then be subjected to downstream purification usingappropriate techniques known in the art. Preferably, the 69 kD proteinis purified using a combination of ion-exchange, hydrophobic interactionand size-exclusion chromatography. Suitable chromatographic supportsinclude anion-exchange such as DEAE-Sepharose, Q-Sepharose,SP-Sepharose, CM-Sepharose; hydrophobic interaction such as Butyl-,Phenyl-, Octyl-Sepharose, TSK; and gel filtration such as Sephacryl,Sepharose, Sephadex, Superose, Superdex, etc.

The highly-purified 69 kD protein may be sterilised by filtration and,if necessary subjected to diafiltration.

The antigenic identity of purified 69 kD outer membrane protein isolatedaccording to the above-described process may be determined by techniqueswell known in the art, for example using SDS-PAGE (Sodium DodecylSulphate-Poly Acrylamide Gel Electrophoresis) and Western blot analysisusing a monoclonal antibody. Purity may be qualitatively assessed bySDS-PAGE. Quantitative analysis may be carried out using ELISA (EnzymeLinked Immuno Sorbent Assay) or HPLC.

Purified 69 kD protein produced according to the invention has beenshown by ELISA testing to have PT and FHA content below the sensitivitylimit of the test. No PT activity (CHO cells) was detectable. Endotoxinlevels are less than 0.1 units/mcg protein by the LAL-CS (LimulusAmoebocyte Lysate-Chromogenic Substrate) test. The 69 kD protein has nodetectable adenylate cyclase activity and the Western blot analysis foradenylate cyclase is negative.

The resulting 69 kD protein has utility as an antigenic component of anacellular vaccine which may be administered to elicit a protectiveantibody response against infection by B. pertussis. Such a vaccine maybe prepared by conventional techniques. For example, a vaccine may beprepared comprising antigenic factors and a suitable conventionalcarrier in an aqueous solution buffered to physiological pH for directuse. Alternatively, antigenic factors may be adsorbed onto aconventional adjuvant, such as aluminium hydroxide or aluminiumphosphate. In addition, one or more B. pertussis antigens, including the69 kD protein, may be combined with further immunogens to preparemulti-functional vaccines, capable of inducing protection against morethan one pathogen.

The present invention has particular utility as part of an overallprocess for the isolation and purification of several protein antigencandidates for an acellular pertussis vaccine from a single fermentationof B. pertussis. Thus PT, FHA and 69 kD protein can each be isolated inpure form from the same fermentation broth for incorporation in a singlepertussis vaccine. The B. pertussis fermentation broth is suitablyprocessed according to the method described in EP-A-0 427 466 to isolatePT and FHA, and the residual supernatant is processed as hereindescribed to provide 69 kD protein. Pertussis toxin may be toxoidedaccording to the method described in WO 91/12020 which is incorporatedherein by reference. The antigen components which have been purifiedseparately may be adjuvanted separately and subsequently pooled.

The extraction of cell-bound protein and the removal of endotoxins fromsolution by addition a flocculating agent, preferably a divalent cation,is not limited to the isolation of outer membrane protein, such aspertactin, from pathogenic Bordetella organisms, such as Bordetellapertussis. The process of the invention has utility in any biologicalprocess involving the production of prophylactic or therapeutic outermembrane proteins. Typical examples of other biological processes whichmay benefit from the invention are:

a) the production of outer membrane proteins from meningitidis speciessuch as Neisseria meningitidis;

b) the production of pili and agglutinogens from Escherichia coli;

c) the production of outer membrane proteins from haemophilus speciessuch as Haemophilus influenzae,

d) the production of outer membrane proteins from borrelia species suchas Borrelia burgdorferi, and

e) the production of outer membrane proteins from streptococcus speciessuch as strains of Group A and Group B streptococcus.

Biological products obtainable according to the process of the inventionare not restricted to proteins for human use. Typical examples offurther processes which may derive benefit are the isolation of outermembrane proteins for use in:

f) the production of vaccines against rhinitis, which affects pigs andis caused by Bordetella bronchiseptica and Haemophilus pleuropneumoniae;

g) the production of a vaccine against colibacillosis, which affectspigs and is caused by Escherichia coli; and

h) the production of an antigen for an ELISA test against leptospirosiswhich affects domestic animals, calves and humans.

The process of the invention is illustrated, but not limited, by thefollowing Examples which relate to the extraction of the 69 kD outermembrane protein from Bordetella pertussis.

In the accompanying drawings:

FIG. 1 shows the results (SDS-PAGE) for the flocculation trials of fourflocculating agents tested in Example 1 against purified 69 kD protein;

FIG. 2 shows the results (Western blot) for the flocculation trials offour flocculating agents tested in Example 1 against purified 69 kDprotein;

FIG. 3 shows the results (SDS-PAGE) for the flocculation trials of threeflocculating agents tested in Example 2 against purified 69 kD proteinand a non-flocculated control;

FIG. 4 shows the results (Western blot) for the flocculation trials ofthree flocculating agents tested in Example 2 against purified 69 kDprotein and a non-flocculated control;

FIG. 5 shows the results (SDS-PAGE) for the flocculation trials withbarium chloride described in Example 3; and

FIG. 6 shows the results (Western blot) for the flocculation trials withbarium chloride described in Example 3.

EXAMPLE 1

Flocculation Trials

A strain of B. pertussis Tohama was cultured under controlled fermenterconditions to provide a suspension of cells of sufficient quantity toextract PT and FHA from the supernatant by adsorption on hydroxyapatitegel. The remaining cell suspension was subjected to the following steps:

1) The suspension was cooled to 4° C., and divided into 50 mL samplealiquots.

2) One of each of the following flocculants was added to each sample togive a final concentration as indicated: dextran T500 (10 g/L), calciumchloride (10 g/L), methanol (20%), and polyethylene glycol 50000 (10g/L).

3) Each sample was vigorously mixed whilst the pH was adjusted to apre-determined value (dextran T500, pH 5; calcium chloride, pH 4 and 9;methanol, pH 6; polyethylene glycol 50000, pH 5). The samples were thenleft to flocculate without mixing at 4° C.

4) The supernatants were decanted off and replaced with equal volumes ofTRIS buffer at a concentration of 125 mM. After resuspension, thesamples were heated to, and maintained at 60° C. in a water bath for aduration of 1 hour. Each sample was shaken regularly (every 5 minutes)to ensure an even temperature distribution.

5) After cooling to 4° C. in the absence of agitation, the dead cellsre-flocculated. After centrifugation, the supernatant from each samplewas tested, not only for the presence of 69 kD protein by SDS-PAGE,Western blot and ELISA, but also for the presence of contaminatingendotoxins using the LAL-CS test.

The results of the SDS-PAGE and the Western blot are given in FIGS. 1and 2, respectively. Each sample (corresponding to one flocculant) wasloaded onto the gel and the blot using two adjacent lanes, correspondingto the volumes 15 and 45 μL. The remaining three lanes were loaded withsuccessively-increasing volumes (6.8, 13.6, and 27.2 μL) from a pool ofpurified 69 kD protein at a concentration of 55 mg/L. The legend for thelanes in the gel and the Western blot can be seen in the followingtable:

                  TABLE 1                                                         ______________________________________                                        Lane                                  Volume loaded                           No     Sample No                                                                              Flocculant                                                                              Concentration                                                                          pH  (μL)                                ______________________________________                                        1    1         Dextran   10 g/L   5   15                                                                            45                                      3           2       CaCl.sub.2                                                                           10 g/L         15                                  4                                                                                                                   45                                      5           3       CaCl.sub.2                                                                           10 g/L         15                                  6                                                                                                                   45                                      7           4       MeOH        20%                                                                                     15 6                                8                                                                                                                   45                                      9           5        PEG        10 g/L                                                                                  15                                  10                                                                                                                  45                                      11         69kD pool                                                                                                                6.8                     12                                                                                                                  13.6                                    13                                                                                                                  27.2                                    ______________________________________                                    

The results of the ELISA and the LAL-CS tests can be seen in thefollowing table:

                  TABLE 2                                                         ______________________________________                                        Sample                          ELISA  Endotoxin                              No      Flocculant                                                                             Concentration                                                                            pH  (mg/L)  (mg/L)                                ______________________________________                                        1      Dextran  10 g/L      5   24.3   >15                                    2         CaCl.sub.2                                                                            10 g/L        4                                                                               22.8    >15                                 3         CaCl.sub.2                                                                            10 g/L        9                                                                               20.3    <15                                 4         MeOH         20%        27.5    >15                                 5         PEG           10 g/L                                                                                5                                                                               31.1    >15                                 ______________________________________                                    

FIGS. 1 and 2 show the presence and location of the 69 kD protein in themajority of samples. The presence of 69 kD protein is confirmed by theresult from the ELISA test shown in table 2 above.

Although flocculation occurred with CaCl₂ at both pH 4 and 9, lanes 5and 6 on the gel (FIG. 1) show a particularly striking example of thereduced level of endotoxins present in the supernatant after treatmentwith CaCl₂ at pH 9. This was confirmed by the results from the LAL-CStest shown in table 2 above.

EXAMPLE 2

Choice of Divalent Cation

Flocculation trials using the methodology described in Example 1 werecarried out using the following halide salts at a concentration of 10g/L and a pH of 9: calcium chloride, barium chloride and strontiumchloride.

The results of the SDS-PAGE and the Western blot are given in FIGS. 3and 4 respectively. The first three lanes were loaded withsuccessively-increasing volumes (6.8, 13.6, and 27.2 μL) from a pool ofpurified 69 kD protein at a concentration of 55 mg/L. The last lane wasloaded with a non-flocculated control sample of the cell suspension. Thelegend for the lanes in the gel and the Western blot can be seen in thefollowing table:

                  TABLE 3                                                         ______________________________________                                        Lane                          Volume loaded                                   No        Sample No    Flocculant                                                                             (μL)                                       ______________________________________                                        1      69kD pool              6.8                                             2                                                   13.6                      3                                                   27.2                      4         1              CaCl.sub.2                                                                                 45                                      5         2              BaCl.sub.2                                                                                 45                                      6         3              SrCl.sub.2                                                                                 45                                      7         4               Control                                                                                    45                                     ______________________________________                                    

The results of the ELISA test can be seen in the following table:

                  TABLE 4                                                         ______________________________________                                        Sample No      Flocculant                                                                             ELISA (mg/L)                                          ______________________________________                                        1              CaCl.sub.2                                                                             15.1                                                  2                     BaCl.sub.2                                                                             15.8                                           3                     SrCl.sub.2                                                                             8.6                                            4                     Control                                                                                  13.5                                         ______________________________________                                    

FIGS. 3 and 4 show the presence and location of the 69 kD protein in allof the samples. The 69 kD protein band observed in the control sample(lane 7) was substantially less dense than those bands from sampleswhich were flocculated with divalent cations (lanes 4, 5 and 6),indicating a substantially-lower concentration of 69 kD protein in thesupernatant from a non-flocculated sample.

The low 69 kD protein concentration given in the ELISA test in Table 4gave the impression that strontium was a less effective cation thaneither calcium or barium, but this observation was not reflected ineither the gel (FIG. 3) or the blot (FIG. 4).

However, the 69 kD concentrations observed in the supernatants ofsamples flocculated with either calcium or barium were substantiallyequal in the ELISA test, and gave 69 kD protein bands of similar densityin both the gel and the blot.

The flocculation process, as observed visually, with reference to therate of flocculation and the morphology of the flocs, was slightly moreeffective with barium than with calcium or strontium.

EXAMPLE 3

Flocculation and Washing Procedure

Flocculation and washing was carried out using the following procedure:

1) The cell suspension remaining after the extraction of PT and FHA wascooled to 4° C., and divided into four sample aliquots of 50 mL. Onesample was set aside as the control.

2) Barium chloride was added to the three remaining samples to give afinal concentration of 10 g/L, and each suspension was mixed vigorouslywhilst the pH was adjusted to 9 using a suitable base.

3) The suspensions were then left to flocculate without mixing at 4° C.

4) The supernatants were decanted off each pellet, and three differentconcentrations of TRIS buffer (125, 50, or 20 mM) were added to eachpellet (one concentration per pellet) to give a final volume of 50 mL.

5) After resuspension, the three samples were then left to flocculate asecond time without mixing at 4° C.

6) The procedure given in 4) and 5) above was repeated as many times asnecessary to dilute the concentration of barium chloride down toapproximately 0.2 g/L.

7) The control sample was centrifuged at 10,000×g for 1 hour, and thesupernatant was decanted off and replaced with an equal volume of TRISbuffer at a concentration of 125 mM.

8) After a final resuspension in TRIS buffer of the requiredconcentration, all the samples (including the control) were heated to,and maintained at 60° C. in a water bath for a duration of 1 hour. Eachsample was shaken regularly (every 5 minutes) to ensure an eventemperature distribution.

9) After cooling all the samples to 4° C. in the absence of agitation,the dead cells in the samples containing barium chloride re-flocculated,and those in the control sample were re-centrifuged at 10,000×g for 1hour.

10) The supernatant from each sample (including the control) was testedfor the presence of 69 kD protein by SDS-PAGE, Western blot and ELISA.

The results of the SDS-PAGE and the Western blot are given in FIGS. 5and 6, respectively. Each sample was loaded onto the gel and the blotusing two adjacent lanes, corresponding to the volumes 15 and 45 μL. Thelegend for the lanes in the gel and the Western blot can be seen in thefollowing table:

                  TABLE 5                                                         ______________________________________                                                                 Volume                                               Lane No         Samples   loaded (μL)                                      ______________________________________                                        1             Control    15                                                   2                                    45                                       3               TRIS 125 mM                                                                             15                                                  4                                    45                                       5                TRIS 50 mM                                                                                 15                                              6                                    45                                       7                TRIS 20 mM                                                                                 15                                              8                                    45                                       ______________________________________                                    

The results of the ELISA test can be seen in the following table:

                  TABLE 6                                                         ______________________________________                                        Samples        ELISA (mg/L)                                                   ______________________________________                                        Control        30                                                             TRIS 125 mM     22                                                            TRIS 50 mM           24                                                       TRIS 20 mM           33                                                       ______________________________________                                    

FIGS. 5 and 6 show the presence and location of the 69 kD protein in allof the samples. The presence of 69 kD protein was confirmed by theresults from the ELISA test shown in table 6 above.

The presence and similarity in the concentrations of 69 kD protein foundin the supernatants of suspensions subjected to repeated flocculationand washing steps described above, indicated that a major reduction inthe ionic strength of the suspending broth, resulting from theapplication of this technique, did not adversely influence theextraction and the yield of 69 kD protein from B. pertussis.

In addition, the effectiveness of barium chloride as a flocculent atconcentrations as low as approximately 0.2 g/L in the supernatant wasdemonstrated.

What is claimed is:
 1. A process for isolating a cell-bound outermembrane protein of bacterial origin in a microbial cell comprising:(a)contacting a microbial cell suspension comprising a cell bound proteinwith a flocculating agent to form a flocculent mass prior to heating;(b) removing said flocculent mass from said suspension; and (c) heatingsaid flocculent mass to release the protein from the microbial cell. 2.The process of claim 1, wherein said process further comprises the stepof isolating the protein.
 3. The process of claim 1, wherein saidcontacting step and said removing step are repeated.
 4. A process forisolating a cell-bound outer membrane protein of bacterial origin in amicrobial cell comprising:(a) contacting a microbial cell suspensioncomprising a cell bound protein with a flocculating agent to form aflocculent mass prior to heating; and (b) heating said flocculent massto release the protein from the microbial cell.
 5. The process of claim4, wherein said process further comprises the step of isolating theprotein.
 6. The process of claim 4 or 5, wherein the cell-bound proteinis an outer membrane protein of a Bordetella, Haemophilus, Escherichia,Streptococcus or Borrelia species.
 7. The process of claim 6, whereinthe outer membrane protein is the 69 kD protein of Bordetella pertussis.8. The process of claim 4, wherein the flocculating agent is a materialembodying a divalent cation.
 9. The process of claim 8, wherein theflocculating agent is a barium, calcium or strontium salt.
 10. Theprocess of claim 9, wherein the salt is barium chloride.
 11. The processof claim 4, wherein the cell-bound protein is suspended in a supernatantat a pH in the range 4 to
 10. 12. A process as claimed in claim 1wherein the cell-bound protein is an outer membrane protein of aBordetella, Haemophilus, Escherichia, Streptococcus or Borrelia species.13. A process as claimed in claim 1 wherein the outer membrane proteinis the a 69 kD protein of Bordetella pertussis.
 14. A process as claimedin claim 1 wherein the flocculating agent is a material embodying adivalent cation.
 15. A process as claimed in claim 14 wherein theflocculating agent is a barium, calcium or strontium salt.
 16. A processas claimed in claim 15 wherein the salt is a halide.
 17. A process asclaimed in claim 16 wherein the salt is a chloride.
 18. A process asclaimed in claim 17 wherein the salt is barium chloride.
 19. A processas claimed in claim 1 wherein the cell-bound protein is suspended in asupernatant at a pH in the range 4 to 10.